Proteins, such as therapeutic antibodies, are often transported and/or stored for later use. It is important that such proteins preserve the stability and biological activity of the protein under various conditions such as different temperature regimens and mechanical stress.
Certain prior liquid antibody preparations have shown short shelf lives and loss of biological activity of the antibodies resulting from chemical and/or physical instabilities during the transportation and storage. Chemical instability may be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange, and physical instability may be caused by antibody denaturation, aggregation, precipitation or adsorption. Among those, aggregation, deamidation and oxidation are known to be the most common causes of the antibody degradation (Cleland et al., 1993, Critical Reviews in Therapeutic Drug Carrier Systems 10: 307-377).
Nanobodies (as further described herein) are characterized by formation of the antigen binding site by a single variable domain, which does not require interaction with a further domain (e.g. in the form of VH/VL interaction) for antigen recognition. Nanobodies against RANKL that can inhibit osteoclast formation and that could be candidates for further drug development are described in WO 08/142,164. The OPG/RANKL/RANK system has recently been discovered as pivotal regulatory factors in the pathogenesis of bone diseases and disorders like e.g. osteoporosis.
Up to now, most of the single variable domains have been administered intravenously. Intravenous (and intramuscular) injections, however, are generally performed by the physician or by the medical professional staff. Therefore, the patient is expected to visit a surgery or a hospital regularly in order to receive treatment. Besides the discomfort created, the time taken up by this type of application often leads to unsatisfactory compliance by the patient, particularly when the treatment extends over several months. Subcutaneous injection renders the possibility to the patient to self-administer the drug and consequently improve patients' cooperation and compliance. These advantages are even more evident in the case of a long-term therapy, such as the treatment of bone diseases and disorders. Another advantage of the subcutaneous administration lies in a substantially lower complication rate due to possible side effects, such as abscess formation and nerve lesions.
The volume of pharmaceutical formulation that can be administered to a patient subcutaneously, however, is more restricted. Therefore, in order to be able to administer a sufficient dose of active substance to the patient, for subcutaneous administration, a higher concentration of drug substance in the pharmaceutical formulation is required so that the required biological effect can still be obtained. Formulation at high protein concentration however comes with a number of challenges and risks, the biggest risk being the formation of aggregates. Understanding the physico-chemical behavior of the single variable domain at high protein concentration is therefore of key importance before defining the final formulation of the pharmaceutical compound.